J Bacteriol Virol.  2011 Dec;41(4):255-265. 10.4167/jbv.2011.41.4.255.

Purification and Characterization of Helicobacter pylori gamma-Glutamyltranspeptidase

• Song JY
• Choi YJ
• Kim JM
• Kim YR
• Jo JS
• Park JS
• Park HJ
• Song YG
• Lee KH
• Kang HL
• Baik SC
• Youn HS
• Cho MJ
• Rhee KH
• Lee WK

• KMID: 2168653
• DOI: http://doi.org/10.4167/jbv.2011.41.4.255

Abstract

Gamma-glutamyltranspeptidase (GGT) was purified to electrophoretic homogeneity from the cell extract of H. pylori. The purified enzyme consisted of heavy and light subunits with molecular weights of 38 kDa and 21 kDa, respectively. N-terminal amino acid sequence of heavy and light subunits revealed that H. pylori GGT was processed into 3 parts for a signal peptide of 27 amino acid residues, a heavy subunit of 352 residues, and a light subunit of 188 residues during translation. The reaction rate for hydrolysis of gamma-GpNA was 84.4 micromol/min per milligram of protein, and that for the gamma-glutamyl transfer from gamma-GpNA to gly-gly was 23.8 micromol/min per milligram of protein. The apparent Km values of H. pylori GGT for gamma-glutamyl compounds were on the order of 10-3 to 10-4 M and those for acceptor peptides and amino acids were on the order of 10-1 to 10-2 M. The GGT protein kept approximately 80% of the initial enzymatic activity on incubation at 60degrees C for 15 min. The optimum temperature and pH for reactions of both hydrolysis and transpeptidation were 40degrees C and 9.0, respectively. The transpeptidation and hydrolysis reactions catalyzed by H. pylori GGT were strongly inhibited by L-Gln and moderately inhibited by L-Ala, L-Ser, beta-chloro-L-Ala, and L-Glu. These results demonstrated that the biochemical properties of H. pylori GGT are different from those of other bacterial GGTs. Further, H. pylori GGT might degrade glutathione in the gastric mucous layer of humans if the enzyme could be secreted in the bacterial niches.

Keyword

H. pylori; gamma-glutamyltranspeptidase; Glutathione

MeSH Terms

Amino Acid Sequence
Amino Acids
Glutathione
Helicobacter
Helicobacter pylori
Humans
Hydrogen-Ion Concentration
Hydrolysis
Light
Molecular Weight
Peptides
Protein Sorting Signals
Amino Acids
Glutathione
Peptides
Protein Sorting Signals

Figure

• 

  Figure 1. Chromatofocusing pattern of H. pylori GGT. Enzyme fractions collected from a hydroxyapatite column were applied on a Mono P column (1 by 5 cm, Pharmacia) equilibrated with 25 mM ethanolamine-acetic acid buffer (pH 9.4) and eluted with 12 bed volumes of polybuffer 96-acetic acid (tenfold dilution [pH 6.0]) to form a descending linear gradient of pH 9 to 6 in the column. Dashed rectangle bars represented the enzyme activity (unit/m1) of fractions. The active fractions (arrow) eluted at pH 8.2 were pooled.

• 

  Figure 2. SDS-polyacrylamide gel electrophoresis of H. pylori GGT in the purification steps. Approximately 8 μg of each fraction was applied. SDS-PAGE was performed with 12.5% polyacrylamide running gel (0.75 mm thick) and 3% polyacrylamide stacking gel at 15 mA for 2 hrs. After electrophoresis, a portion of the gel was stained with Coomassie brilliant blue R250. The subunit molecular weight was estimated by SDS-PAGE. Protein molecular weight standard, high range marker (200, 97.4, 68, 43, 29, 18.4, and 14.3 kDa) was used as a standard. M, marker; W, cell extract; SO, salt precipitation; GF, gel-filtration fraction; RS, Resource S fraction; HA, hydroxyapatite fraction; CF, chromatofocusing fraction.

• 

  Figure 3. Temperature stability and reactivity of H. pylori GGT at the various conditions of temperature and pH. (A) reaction mixtures were incubated at various temperatures for 15 min in 20 mM Tris-HCl (pH 8.0), and then GGT activities were measured; (B) GGT activities were measured at various temperature on the standard conditions described in Materials and Methods; (C) the pH of the reaction mixture containing the enzyme was adjusted with phosphate buffer for pH 4.0∼6.0 or Tris-HCl buffer for pH 7.0∼11.0 (open circles, transpeptidation activity; closed circles, hydrolysis activities). The indicated value is the mean of two independent experiments.

• 

  Figure 4. Lineweaver-Burk plot of transpeptidation and/or hydrolysis of γ-GpNA, gly-gly, L-Arg, and GSH as catalyzed by purified H. pylori GGT. The enzyme activities were measured on the conditions described in Materials and Methods except for the change of the pH 9.0. (A) donor activities of γ-glutamyl moiety of γ-GpNA in transpeptidation reaction (○) and in hydrolysis reaction (•); (B) acceptor activities of gly-gly (⋄) and L-Arg (△) in transpeptidation reaction; C, donor activities of glutamyl moiety of GSH in transpeptidation reaction (□) and in hydrolysis reaction (▪). The reciprocals of the rates of 2-nitroaniline production (A, B) or γ-glutamyl-glycylglycine and glutamic acid production (C) were plotted against those of different concentrations of γ-glutamyl moiety donors or acceptors. The indicated values are the mean of two independent experiments.

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